The goal of this renewal is to exploit new insights into viral mechanisms that inactivate p53 to develop potent p53 tumor selective oncolytic adenovirus therapies. The p53 pathway is inactivated by mutations in almost every form of human cancer. However, despite this, there are still no approved rational therapies that target p53 defective tumor cells. Adenovirus E1B-55K targets p53 for degradation, which was thought to be essential for p53 inactivation and viral replication. On this premise, a ?B-55K virus, ONYX-015, was tested in clinical trials as a p53 cancer therapy. However, patient responses did not correlate with the p53 status of their tumors. Although p53 is induced, it is inactive. Consequently, p53 does not determine the tumor selective replication of ONYX-015. As such, the enormous potential of a p53 selective could still be realized if the E1B-55K independent mechanisms through which adenovirus inactivates p53 are determined. This was the goal of the previous grant period. This led to the discovery that Ad5 encodes another protein, E4-ORF3, which inactivates p53 by forming a nuclear polymer that specifies de novo heterochromatin silencing of p53 target promoters, thereby preventing p53-DNA binding. With access denied, p53 is powerless to activate the transcription of downstream effectors to limit viral replication. These studies changed the longstanding definition of how p53 is inactivated in adenovirus infection. Here we propose to exploit these mechanistic insights to engineer E1B-55K/E4-ORF3 mutant adenoviruses as exciting new agents for p53 selective oncolytic viral therapy. This requires E1B-55K and E4-ORF3 mutations that selectively uncouple p53 inactivation from their other functions in viral replication. Previous studies revealed an E1B-55K H260A mutation that fulfills these criteria. To identify analogous mutations in E4-ORF3, we solved the atomic structure of Ad5 E4-ORF3. In Aim 1, we will use this to reveal novel structural motifs that silence p53 target genes. We have discovered that E4-ORF3 proteins from other Adenovirus subgroups do not inactivate p53. This provides a rational basis to identify Ad5 E4-ORF3 specific motifs that silence p53 target genes and engineer viruses with discrete E4-ORF3 mutations that prevent p53 inactivation. In Aim 2, we will combine E4ORF3 and E1B-55K mutations to develop novel Ad5 and Ad34 viruses and test their p53 selectivity in panels of normal and tumor cells. These studies will identify the critical functions of p53 that prevent viral replication and if they are disrupted by p53 and p14ARF mutations in cancer. Finally, in Aim 3, we will test novel p53 selective oncolytic viruses in preclinical mouse models of cancer. We will exploit new technologies to engineer additional genomic modifications that prevent viral uptake in the liver and limiting inflammation, enabling systemic delivery and expanded tumor tropisms. These viral therapies have the potential to be self-perpetuating, kill tumors through regulated cell death, and produce progeny that spread from within the tumor to distant micro-metastases. If results in pre-clinical models are promising, the goal is to test these agents in patients.